US20240091742A1 - Components for separating molecules and methods of making and using the same - Google Patents

Components for separating molecules and methods of making and using the same Download PDF

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US20240091742A1
US20240091742A1 US18/229,553 US202318229553A US2024091742A1 US 20240091742 A1 US20240091742 A1 US 20240091742A1 US 202318229553 A US202318229553 A US 202318229553A US 2024091742 A1 US2024091742 A1 US 2024091742A1
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resin
bsa
kda
molecules
size exclusion
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Ramesh Ganapathy
Brendon Wang
Ashok Salunkhe
Deven Etnyre
Barbara Kaboord
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Pierce Biotechnology Inc
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Pierce Biotechnology Inc
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Assigned to PIERCE BIOTECHNOLOGY, INC. reassignment PIERCE BIOTECHNOLOGY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GANAPATHY, RAMASWAMI, WANG, Brendon
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/288—Polar phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3847—Multimodal interactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
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    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography

Definitions

  • the present disclosure concerns a matrix, system, method, and kit for sample preparation, such as separating small molecules from large molecules.
  • Sample preparation techniques for isolating biomolecules may require separating biomolecules from other sample components and from sample processing components to enable downstream analysis and processing of the biomolecule.
  • sample preparation of biomolecules such as proteins or nucleic acids
  • the biomolecules may need to be chemically modified, such as by reduction, oxidization, cross-linking, and/or alkylation.
  • BSA Bovine Serum Albumin
  • FPLC fast protein liquid chromatography
  • Disclosed embodiments of the present disclosure advantageously provide superior separation of molecules, such as biomolecules, from each other.
  • molecules are separated from each other using differences in one or more properties, such as, but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties.
  • molecules can be separated from each other based on one or more separation matrix properties, such as, but not limited to, the charge and size exclusion properties. This can advantageously reduce time and expense related to the separating of small molecules from larger molecules.
  • Molecules separated as set forth herein facilitate downstream processing relative to known processes.
  • the porous size exclusion support is produced by using sufficient HEC to produce a resin with a molecular weight cut-off (or “MWCO”) greater than or equal to 40 kDa.
  • MWCO molecular weight cut-off
  • some embodiments may comprise from 60 grams HEC to 150 grams HEC.
  • 60 grams to 130 grains HEC in a 5 liter reaction scale is used to produce the size exclusion support.
  • 0.5 liters to 2 liters of resin bed. comprising the porous size exclusion support is produced from 60 grams FIEC to 130 grams HEC.
  • the porous size exclusion support may further comprise a crosslinked porous size exclusion support.
  • the porous size exclusion support can be crosslinked with an epoxide-containing compound comprising at least one epoxide functional group, with exemplary embodiments being crosslinked with epichlorohydrin (also referred to herein as “Epi”).
  • Epi epichlorohydrin
  • the porous size exclusion support can be crosslinked with 250 milliliters Epi to 450 milliliters Epi.
  • Certain disclosed embodiments concern a matrix comprising a porous size exclusion support having a molecular weight cutoff of greater than or equal to 40 kDa, and at least one cationic moiety associated with the porous size exclusion support, wherein the at least one cationic moiety is selected for association with a small molecule having a molecular weight less than 100 kDa, such as, but not limited to, Bovine Serum Albumin (BSA), and/or other proteins.
  • BSA Bovine Serum Albumin
  • at least one cationic moiety associates with the small molecule by ionic interaction, hydrophilic interactions, hydrophobic interactions, affinity interaction, hydrogen bonding, and/or van der Waals forces.
  • the cationic moiety may be, for example, an amine, a diamine, a polyamine, amine-containing heterocyclic compounds, amine-containing aromatic compound, or an amine-containing polymer.
  • Particular examples of cationic moieties include 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine, polyethylene imine, diaminopentane, N,N-diethylethylenediamine, 1,2-diaminobenzene, 1,3-diaminobenzene, 1,4-diaminobenzene, and (S)-N-boc -2,3 -epoxypropylamine.
  • the cationic moiety is covalently bound to the porous size exclusion support.
  • the matrix may be advantageously equilibrated with an equilibration buffer, comprising little to no salt (e.g., NaCl and other ionic salts), such as from 0 mM of salt to 5 mM of salt.
  • the equilibration buffer may comprise a positive charge.
  • the equilibration buffer may comprise a neutral charge.
  • the equilibration buffer can have a pH of 4 to a pH of 9, preferably a pH of 5 to pH of 7.
  • the equilibration buffer can have a concentration of from 20 mM to 100 mM.
  • the equilibration buffer may comprise triethylammonium bicarbonate, borate, sodium acetate, or HEPES to facilitate separating a small molecule from a larger molecule. For example, separating a negatively charged small molecule from a positively charged large molecule.
  • the present disclosure also provides a system for separating molecules of varying molecular weights and/or charges and/or isoelectric point (pI) values from each other.
  • a system of the disclosure can separate small molecule from a large molecule in a sample.
  • a system can of the disclosure can separate small molecules, such as proteins of 70 kDa, from large molecules, such as proteins of 150 kDa, from a sample.
  • Such systems comprise a container comprising a matrix of the present disclosure, and a receptacle located to receive flow from the container.
  • the system may be configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or combinations thereof.
  • the container may be any suitable container, such as a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.
  • a method for making a matrix comprising a porous size exclusion support having a MWCO of greater than or equal to 40 kDa, and at least one cationic moiety associated with the porous size exclusion support is also disclosed.
  • the method comprises providing a porous size exclusion support comprising hydroxyethyl cellulose having a MWCO of greater than or equal to 40 kDa or greater, the hydroxyethyl cellulose having at least one vicinal diol.
  • the vicinal diol is oxidized to form an aldehyde, and the aldehyde is then reacted with an amine group of a cationic moiety of at least greater than 50 mg/mL via reductive amination using, for example, sodium cyanoborohydride or picoline borane.
  • concentration of the cationic moiety that is used to form the resin can be varied, as desired, to facilitate separation processes, but typically is within a concentration of 50 mg/mL to 175 mg/mL, more typically 50 mg/mL to 160 mg/mL.
  • a method of the disclosure can separate a protein of less than or equal to 70 kDa from at least one large molecule of greater than 100 kDa in a sample.
  • the method comprises providing a matrix comprising a porous size exclusion support having at least one cationic moiety associated therewith, wherein the cationic moiety can associate with at least one small molecule, such as but not limited to, a negatively charged small molecule.
  • the matrix can be equilibrated with an equilibration buffer.
  • a sample is then applied to the matrix to separate the at least one small molecule from the at least one large molecule by subjecting the matrix to gravity flow, a centrifugal force, a positive pressure, a negative pressure, a vacuum, or a combination thereof.
  • At least one large molecule in the sample is excluded by the matrix and is collected as a flow-through in a receptacle located to receive the flow-through.
  • the at least one small molecule e.g., a negatively charge small molecule
  • the disclosed method substantially increases the ability to separate small molecules, such as Bovine Serum Albumin, from the sample, and also substantially increases the recovery of the large molecule, such as IgG. This in turn facilitates processing the large molecule in downstream applications, such as dye labeling.
  • kits of the disclosure can separate small molecules, such as proteins of 70 kDa, from large molecules, such as proteins as large as 150 kDa, from a sample.
  • the kit of the disclosure can separate negatively charged molecules from more positively charged molecules in a sample.
  • the kit may comprise (1) a porous size exclusion support having at least one cationic moiety associated therewith, wherein the cationic moiety can associate with and capture the small molecule, and (2) instructions for using the porous size exclusion support.
  • the kit may further comprise an equilibration buffer, and/or a system comprising a container housing the porous size exclusion support, and a receptacle positioned to receive flow-through the support.
  • FIG. 1 is a schematic drawing illustrating one disclosed embodiment comprising associating a porous size exclusion support with a cationic moiety, such as diaminopentane (or “PDA”).
  • a cationic moiety such as diaminopentane (or “PDA”).
  • FIG. 2 is a schematic drawing illustrating one disclosed embodiment comprising associating a porous size exclusion support with a cationic moiety, such as a branched polyethylene imine (or “PEI”).
  • a cationic moiety such as a branched polyethylene imine (or “PEI”).
  • FIG. 3 is a schematic drawing illustrating one disclosed embodiment comprising associating a porous size exclusion support with a cationic moiety, such as N,N diethylethylenediamine (or “DEED”).
  • a cationic moiety such as N,N diethylethylenediamine (or “DEED”).
  • FIG. 4 is a schematic drawing illustrating one disclosed embodiment of using a cationic moiety associating onto a porous size exclusion support to separate a small molecule from a sample comprising the small molecule such as but not limited to, a negatively charged small molecule.
  • FIG. 5 is a schematic drawing illustrating one embodiment of a disclosed system comprising a container, receptacle, and an exemplary matrix comprising a porous size exclusion support associated with a cationic moiety.
  • FIG. 6 is a schematic side view of a one embodiment of a system according to the present disclosure comprising a container and a receptacle for processing a sample to separate at least one small molecule from at least one large molecule using differences in one or more properties, such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties.
  • a container and a receptacle for processing a sample to separate at least one small molecule from at least one large molecule using differences in one or more properties, such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties.
  • pI isoelectric point
  • FIG. 7 is a schematic perspective view of one embodiment of a disclosed system for processing a sample to separate a small molecule from a large molecule using differences in one or more properties, such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties, and wherein the system comprises a multi-well container and a receptacle.
  • properties such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties, and wherein the system comprises a multi-well container and a receptacle.
  • FIG. 8 is a schematic perspective view of one embodiment of a disclosed system for processing a sample to separate a small molecule from a large molecule using differences in one or more properties, such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties, and wherein the system comprises a multi-well container and a receptacle.
  • properties such as but not limited to, the size of the molecules, charge of the molecules, the isoelectric point (pI) of molecules, and/or any combination of these properties, and wherein the system comprises a multi-well container and a receptacle.
  • FIG. 9 is an image of a gel comparing the BSA removal and IgG recovery of the following embodiments disclosed herein at Table 2 from a sample comprising a mixture of BSA (2 mg/mL) and IgG (2 mg/mL): Resin A, Resin B, Resin C, Resin D, Resin E, and Resin F; wherein FIG. 9 shows that the greatest to least BSA removal was achieved in the following order (and thus shows that in addition to the PDA, the MWCO also contributed to the desired BSA removal): Resin F, Resin D, Resin E, Resin B, then Resin A.
  • FIG. 10 is an image of a gel comparing the Resin 5 embodiment (as described in Table 1, provided herein), the Resin L embodiment (as described in Table 2, provided herein), the Resin F embodiment (as described in Table 2, provided herein), and the Resin D embodiment (as described in Table 2, provided herein) were equilibrated with different equilibration buffers (as described in Table 3, provided herein) to remove BSA and recover IgG from a sample comprising a mixture of BSA (10 mg/mL) and IgG (1 mg/mL); thus, this figure demonstrates that PDA helps remove a desirable amount of BSA, the embodiments having a MWCO of greater than or equal to 40 kDa exhibited a lower capacity for removing BSA than the 45 kDa MWCO embodiments, and the embodiments equilibrated with Tris buffer showed a desirable IgG recovery.
  • FIG. 11 is an image of a gel showing the removal of BSA and recovery of IgG from a
  • sample comprising a mixture of BSA (10 mg/mL) and IgG (1 mg/mL) by the Resin G embodiment (as described in Table 1, provided herein) equilibrated with 50 mM Tris pH 7 (Lane 1), 50 mM TEAB pH 5 (Lane 2), 50 mM TEAB pH 7 (Lane 3), 50 mM sodium acetate pH 5 (Lane 4), 50 mM sodium acetate pH 7 (Lane 5), 50 mM HEPES pH 5 (Lane 6), 50 mM HEPES pH 7 (Lane 7) showing desirable BSA removal and IgG recovery from the sample.
  • FIG. 12 is a bar graph comparing the BSA binding capacity (300 ⁇ L of 10 mg/mL BSA) of the Resin 5 embodiment (as described in Table 1, provided herein) modified with 150 mg/mL PEI, the Resin 3 embodiment (as described in Table 1, provided herein) modified with 130 mg/mL PEI , MelonTM Gel IgG Purification Kit, and Affi-Gel® Blue Gel (Bio-Rad); the Resin 5 embodiment exhibited a binding capacity of 3.11 mg BSA bound/mL resin, the Resin 3 embodiment exhibited a binding capacity of 2.45 mg BSA bound/mL resin, the MelonTM Gel IgG Purification Kit exhibited a binding capacity of 1.26 mg BSA bound/mL resin, and the Affi-Gel® Blue Gel (Bio-Rad) exhibited a binding capacity of 2.43 mg BSA bound/mL resin; thus, this figure shows a desirable BSA binding capacity in the Resin 5 embodiment modified with 150 mg/mL PEI,
  • FIG. 13 is a bar graph comparing the IgG recovery (2 mg/mL) of the Resin 5 embodiment (as described in Table 1, provided herein) modified with 150 mg/mL PEI, the Resin 3 embodiment (as described in Table 1, provided herein) modified with 130 mg/mL PEI, MelonTM Gel IgG Purification Kit, and Affi-Gel® Blue Gel (Bio-Rad); the Resin 5 embodiment exhibited a 72% volume recovery, the Resin 3 embodiment exhibited a 61% volume recovery, the MelonTM Gel IgG Purification Kit exhibited a 80% volume recovery, and the Affi-Gel® Blue Gel (Bio-Rad) exhibited a 25% volume recovery; thus, this example shows a desirable IgG recovery in the Resin 5 embodiment modified with 150 mg/mL PEI, the Resin 3 embodiment modified with 130 mg/mL PEI, and the MelonTM Gel IgG Purification Kit unlike the Affi-Gel® Blue Gel (Bio-Rad
  • FIG. 14 A is an image of a gel comparing the Resin 5 embodiment (as described in Table 1, provided herein) produced with different amounts of PDA (as described in Table 4, provided herein) to Abcam BSA Removal Kit and the MelonTM Gel IgG Purification Kit to remove BSA and recover IgG from a sample comprising a mixture of BSA (10 mg/mL) and GAR (1 mg/mL); the Resin 5 embodiment modified with 150 mg/mL PDA (also referred to herein as the Resin F embodiment as described in Table 2, provided herein) and the Resin 5 embodiment modified with 75 mg/mL PDA (also referred to herein as the Resin K embodiment as described in Table 2, provided herein) showing higher BSA removal and higher recovery of IgG than Abcam BSA Removal Kit and MelonTM Gel IgG Purification Kit.
  • PDA as described in Table 4, provided herein
  • FIG. 14 B is a bar graph showing the band quantification of the gel of FIG. 14 A using iBright Image analysis software demonstrating the Resin 5 embodiment modified with 150 mg/mL PDA had a 85.7% IgG recovery and a 98.8% BSA removal; Resin 5 modified with 75 mg/mL showed an 86.7% IgG recovery and an 82.6% BSA removal; Abcam BSA Removal Kit had a 53.3% IgG recovery and a 65.78% BSA removal; and MelonTM Gel IgG Purification Kit had a 78.7% IgG recovery and a 8.3% BSA removal; thus, demonstrating the Resin 5 modified with 150 mg/mL PDA and 75 mg/mL PDA achieved a higher IgG recovery and BSA removal than the Abcam BSA Removal Kit and the MelonTM Gel IgG Purification Kit.
  • FIG. 15 is a bar graph showing the percent recovery of different sized molecules to establish a MWCO of the Resin 5 embodiment, the Resin 6 embodiment, the Resin 7 embodiment, and the Resin 8 embodiment (as described in Table 1, provided herein) showing the Resin 5 embodiment exhibited an 86% at 42,000 Da, 94% recovery at 67,000 Da, 92% recovery at 80,000 Da, and 94% recovery at 150,000 Da; the Resin 6 embodiment exhibited an 82% recovery at 42,000 Da, 84% recovery at 67,000 Da, 90% recovery at 80,000 Da, and 92% recovery at 150,000 Da; the Resin 7 embodiment exhibited a 66% recovery at 42,000 Da, 76% recovery at 67,000 Da, 84% recovery at 80,000 Da, and 86% recovery at 150,000 Da; the Resin 8 embodiment exhibited a 58% recovery at 42,000 Da, 75% recovery at 67,000 Da, 75% recovery at 80,000 Da, and 83% recovery at 150,000 Da; thus, this figure demonstrates that by decreasing the amount of HEC, resulted in resins with a 50 kDa
  • FIG. 16 A is an image of a gel showing the removal of BSA and IgG recovery for the Resin F embodiment, the Resin G embodiment, the Resin H embodiment, the Resin I embodiment, and the Resin J embodiment (as described in Table 1, provided herein) versus MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit from a sample comprising a mixture of BSA (10 mg/mL) and GAR IgG (1 mg/mL); thus, this figure demonstrates the Resin F embodiment and the Resin G embodiment had the most desirable BSA removal and IgG recovery from the sample.
  • FIG. 16 B a bar graph showing the band quantification of the gel of FIG. 16 A using iBright Image analysis software demonstrating the that Resin F embodiment had a 83% GAR recovery and a 99% BSA removal; the Resin G embodiment had a 93% GAR recovery and a 100% BSA removal; the Resin H embodiment had a 63% GAR recovery and a 99% BSA removal; the Resin I embodiment had a 76% GAR recovery and a 95% BSA removal; the Resin J embodiment had a 82% GAR recovery and a 100% BSA removal; MelonTM Gel IgG Purification Kit (Thermo ScientificTM) had a 106% GAR recovery and a 82% BSA removal; Affi-Gel® Blue Gel (Bio-Rad) had a 73% GAR recovery and a 65% BSA removal; Abcam BSA Removal Kit had a 125% GAR recovery and a 85% BSA removal; thus, this figure demonstrates higher BSA removal and IgG recovery by the Resin F embodiment
  • FIG. 17 is am image of a gel showing the BSA removal and IgG recovery of the Resin F embodiment, the Resin G embodiment, the Resin H embodiment, the Resin I embodiment, and the Resin J embodiment (as described in Table 2, provided herein) versus the MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit for a sample comprising a mixture of BSA (10 mg/mL) and IgG (0.1 mg/mL); thus, this figure demonstrates that the Resin F embodiment, the Resin G embodiment, and the Resin J embodiment performed better in mixture having a low concentration of IgG versus the MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel(Bio-Rad), and Abcam BSA Removal Kit.
  • FIG. 18 is an image of a fluorescently labeled GAR flow-through with NHS DyLightTM 488 (Thermo ScientificTM) after removing BSA from a mixture comprising GAR (1 mg/mL) and BSA (10 mg/mL) with the Resin F embodiment (as described in Table 2, provided herein) versus MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit; thus, this figure demonstrates the desirable ability to recovery the labeled GAR after the BSA removal by the Resin F embodiment and Abcam BSA Removal Kit unlike MelonTM Gel IgG Purification Kit (Thermo ScientificTM) and the Affi-Gel® Blue Gel (Bio-Rad).
  • FIG. 19 is an image of a fluorescently labeled GAR flow-through with NHS DyLightTM 488 (Thermo ScientificTM) after removing BSA from a mixture comprising GAR (0.1 mg/mL) and BSA (10 mg/mL) with the Resin F embodiment and the Resin G embodiment (as described in Table 2, provided herein) versus MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit; thus, this figure demonstrates the desirable ability to recover the labeled GAR after the BSA removal by the Resin F embodiment and Resin G embodiment even at low antibody concentrations, such as 0.1 mg/mL unlike MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit.
  • NHS DyLightTM 488 Thermo ScientificTM
  • FIG. 20 is an image of a gel showing the cleanup of rabbit serum, mouse serum, human plasma, and human serum by the Resin F embodiment and Resin G embodiment (as described in Table 2, provided herein) and demonstrating desirable removal of albumin and desirable IgG recovery from different serum species.
  • FIG. 21 A is a bar graph showing the rabbit IgG (A 280 amount) for the Resin G embodiment (as described in Table 2, provided herein) equilibrated with different concentrations of Tris buffer showing the A 280 amount of the rabbit IgG of the Resin G embodiment equilibrated with 50 mM Tris (pH 7.0) had a 123 A 280 amount; the Resin G embodiment equilibrated with 50 mM Tris (pH 5.0) had a 124 A 280 amount; the Resin G embodiment equilibrated with 50 m Tris (pH 7.0 +stacker 20 ⁇ L) had a 139 A 280 amount; the BSA-rabbit IgG start mixture had a 824 A 280 amount; the Rabbit IgG start had a 133 A 280 amount; and the BSA only had a 634 A 280 amount; thus, the figure demonstrates the desirable recovery of the antibody because the flow-through A 280 amount was similar to the A 280 amount of the rabbit
  • FIG. 21 B is a bar graph showing the GAR (A 280 amount) for the Resin G embodiment (as described in Table 2, provided herein) equilibrated with different concentrations of Tris buffer showing the A 280 amount of the rabbit IgG of the Resin G embodiment equilibrated with 50 mM Tris (pH 7.0) had a 97 A 280 amount; the Resin G embodiment equilibrated with 50 mM Tris (pH 5.0) had a 101 A 280 amount; the resin G embodiment equilibrated with 50 m Tris (pH 7.0+ stacker 20 ⁇ L) had a 99 A 280 amount; the BSA-GAR start mixture had a 834 A 280 amount; the GAR start had a 100 A 280 amount; and the BSA only had a 634 A 280 amount; thus, this figure demonstrates the figure demonstrates the desirable recovery of the antibody because the flow-through A 280 amount was similar to the A 280 amount of the GAR.
  • FIG. 22 is an image showing the labeling of primary antibody (GAPDH) with fluorescent dye after BSA removal from sample comprising a mixture of antibody (1 mg/mL)-BSA (10 mg/mL) for the Resin G embodiment (as described in Table 2, provided herein) spun at 3,000 ⁇ G and at 6,000 ⁇ G, the antibody-BSA and free Dy 650 was also labeled; thus this figure shows a desirable BSA removal for both spin speeds.
  • GPDH primary antibody
  • FIG. 22 is an image showing the labeling of primary antibody (GAPDH) with fluorescent dye after BSA removal from sample comprising a mixture of antibody (1 mg/mL)-BSA (10 mg/mL) for the Resin G embodiment (as described in Table 2, provided herein) spun at 3,000 ⁇ G and at 6,000 ⁇ G, the antibody-BSA and free Dy 650 was also labeled; thus this figure shows a desirable BSA removal for both spin speeds.
  • FIG. 23 is an image a gel obtained by loading and staining the flow-throughs using Coomassie stain using Pierce Power blotter showing the Calreticulin that is free from BSA after passing through the Resin G embodiment (as described in Table 2, provided herein), the Calreticulin that is conjugated to the DyLightTM 680 (Thermo ScientificTM) after BSA removal, and the Calreticulin as received with BSA added as a stabilizer; thus, this figure demonstrates the desirable removal of BSA from the primary antibody Calreticulin before conjugating it with DyLightTM (Thermo ScientificTM) 680 by the Resin G embodiment.
  • FIG. 24 A is an image of Western Blot application using fluorescent labeled GAR after BSA cleanup with the Resin H embodiment (as described in Table 2, provided herein) showing the BSA removed from GAR (left) and the BSA not removed from GAR (right) from a sample comprising a mixture of GAR (1 mg/mL) and BSA (10 mg/mL); thus, this figure shows that BSA removed from the GAR before conjugating to Dy 650 showed a much higher intensity than when BSA was not removed from the GAR.
  • FIG. 24 B is a bar graph showing the fluorescence intensity of the removed BSA with the Resin H embodiment (as described in Table 2, provided herein) and the unremoved BSA of a HeLa lysate load for the BSA removed at 10 ⁇ g had a fluorescence intensity of 13,000,000 and the unremoved BSA had a fluorescence intensity of 4,000,000; HeLa lysate load for the BSA removed at 5 ⁇ g had a fluorescence intensity of 9,000,000 and the unremoved BSA had a fluorescence intensity of 3,800,000; HeLa lysate load for the BSA removed at 2.5 ⁇ g had a fluorescence intensity of 7,800,000 and the unremoved BSA had a fluorescence intensity of 3,800,000; and HeLa lysate load for the BSA removed at 1.25 ⁇ g had a fluorescence intensity of 4,100,000 and the unremoved BSA had a fluorescence intensity of 1,800,000; thus, this figure
  • BSA Bovine Serum Albumin
  • HEC hydroxyethyl cellulose
  • IgG Immunoglobulin G
  • PEI Branched polyethylene imine
  • values, procedures, or devices may be referred to as “lowest,” “best,” “minimum,” or the like. It will be appreciated that such descriptions are intended to indicate that a selection among many used functional alternatives can be made, and such selections need not be better, smaller, or otherwise preferable to other selections.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof” is intended to include at least: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • acyl halide generally refers to —C(O)X, wherein X is a halogen, such as Br, F, I, or Cl.
  • Alcohol generally refers to an organic compound including at least one hydroxyl group.
  • Alcohols may be monohydric (including one —OH group), dihydric (including two —OH groups; diols, such as glycols), trihydric (including three —OH; triols, such as glycerol) groups, or polyhydric (including two or more —OH groups; polyols).
  • the organic portion of the alcohol may be aliphatic, cycloaliphatic (alicyclic), heteroaliphatic, cycloheteroaliphatic (heterocyclic), polycyclic, aryl, or heteroaryl, and may be substituted or unsubstituted.
  • aldehyde generally refers to generally refers to a carbonyl-bearing functional group having a formula
  • aliphatic generally refers to a substantially hydrocarbon-based compound, or a radical thereof (e.g., C 6 H 13 , for a hexane radical), including alkanes, alkenes, alkynes, including cyclic versions thereof, and further including straight- and branched-chain arrangements, and all stereo and position isomers as well. Unless expressly stated otherwise, an aliphatic group contains from one to twenty-five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms. The term “lower aliphatic” refers to an aliphatic group containing from one to ten carbon atoms.
  • An aliphatic chain may be substituted or unsubstituted. Unless expressly referred to as an “unsubstituted aliphatic,” an aliphatic group can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a C ⁇ C double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group).
  • substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality.
  • alkoxy generally refers to radical (or substituent) having the structure —OR, where R is a substituted or unsubstituted alkyl.
  • alkyl generally refers to a hydrocarbon group having a saturated carbon chain.
  • the chain may be cyclic, branched, or unbranched.
  • alkynyl generally refers to an organic compound having at least one carbon-carbon triple bond.
  • An alkynyl group can be branched, straight-chain, or cyclic (e.g., cycloalkynyl).
  • amide generally refers to chemical functional group —C(O)N(R′)(R′′) where R′ and R′′ independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • amino generally refers to a chemical functional group —N(R)R′ where R and R′ are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • antibody generally refers to immunoglobulins or immunoglobulin-like molecules (including by way of example and without limitation, IgA (Immunoglobulin A), IgD (Immunoglobulin D), IgE (Immunoglobulin E), IgG (Immunoglobulin E) and IgM (Immunoglobulin M), combinations thereof, and similar molecules produced during an immune response in any chordate such as a vertebrate, for example, in mammals such as humans, goats, rabbits and mice) and fragments thereof that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules.
  • immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA (Immunoglobulin A), IgD (Immunoglobulin D), IgE (Immunoglobulin E), IgG (Immunoglobulin E) and IgM (I
  • an “antibody” typically comprises a polypeptide ligand having at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of an antigen.
  • Immunoglobulins are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V H ) region and the variable light (V L ) region. Together, the V H region and the V L region are responsible for binding the antigen recognized by the immunoglobulin.
  • immunoglobulin fragments include, without limitation, proteolytic immunoglobulin fragments [such as F(ab′) 2 fragments, Fab′ fragments, Fab′-SH fragments and Fab fragments as are known in the art], recombinant immunoglobulin fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, ‘F(ab)’ 2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”).
  • proteolytic immunoglobulin fragments such as F(ab′) 2 fragments, Fab′ fragments, Fab′-SH fragments and Fab fragments as are known in the art
  • recombinant immunoglobulin fragments such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, ‘F(ab)’ 2 fragments, single chain
  • Antibody also includes genetically engineered molecules, such as chimeric antibodies (for example, humanized murine antibodies), and heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
  • aromatic generally refers to a cyclic or conjugated group comprising, unless specified otherwise, from 5 to 15 ring atoms having at least a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring, and optionally multiple condensed rings, have a continuous, delocalized ⁇ -electron system.
  • the number of out of plane ⁇ -electrons corresponds to the Hiickel rule (4n+2).
  • the point of attachment to the parent structure typically is through an aromatic portion of the condensed ring system.
  • context or express disclosure may indicate that the point of attachment is through a non-aromatic portion of the condensed ring system.
  • Aromatic group may comprise only carbon atoms in the ring, such as in an aryl group, or it may comprise one or more ring carbon atoms and one or more ring heteroatoms comprising a lone pair of electrons (e.g. S, O, N, P, or Si), such as in a heteroaryl group.
  • Aromatic groups may be substituted with one or more groups other than hydrogen, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality , or an organic functional group.
  • aryl generally refers to an aromatic carbocyclic group comprising at least five carbon atoms to 15 carbon atoms (C 5 -C 15 ), such as five to ten carbon atoms (C 5 -C 10 ), having a single ring or multiple condensed rings, which condensed rings can or may not be aromatic provided that the point of attachment to a remaining position of the compounds disclosed herein is through an atom of the aromatic carbocyclic group.
  • Aryl groups may be substituted with one or more groups other than hydrogen, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • groups other than hydrogen such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • biological sample generally refers to hematological, cytological and histological specimens, such as cells, cell cultures, hybridomas, single-celled organisms (e.g. yeast and bacteria), 3D cell cultures (e.g. spheroids and organoids), tissues, whole organisms (e.g. flies or worms), cell-free extracts, or a fluid sample comprising any biological matter (e.g., blood, serum, plasma, sputum, urine, cerebrospinal fluid).
  • Biological samples can be from a plant or animal (e.g., human, mouse, fly, worm, fish, frog, fungi, and the like).
  • a sample can refer to a sample that has been processed by filtration and/or centrifugation and can include supernatants of cell cultures and homogenized tissue or broken up cells.
  • carboxylate generally refers to —OC(O)NRR′, wherein R and R′ independently are hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • carbonate generally refers to a functional group with the formula —OCO 2 R where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • Carboxamide generally refers to the —N(R)acyl, or —C(O)amino, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • carboxyl generally refers to —C(O)OH.
  • carboxylic acid generally refers to an organic compound having a formula RCOOH where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • cyano generally refers to —CN.
  • diisulfide generally refers to —SSR a , wherein R a is selected from hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • epoxy generally refers to a cyclic ether with a 3-membered ring having a general formula
  • R 1 -R 4 independently are hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • esters generally refers to a chemical compound having a formula
  • R and R′ are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • ether generally refers to a class of organic compounds containing an ether group, that is an oxygen atom connected to two aliphatic and/or aryl groups and having a general formula R—O—R′, where R and R′ are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • equilibrium buffer generally refers to a buffer that is used to infuse a matrix according to the present disclosure to facilitate processing a sample and to promote the affinity of a molecule of interest to the support.
  • fluorophore generally refers to a functional group or portion of a compound that causes the compound (or a sample or composition comprising the compound), to fluoresce.
  • the fluorophore can fluoresce when the compound (or a sample or composition comprising the compound) is exposed to an excitation source or after being cleaved from a compound to which the fluorophore is conjugated.
  • the term “functional group” generally refers to a specific group of atoms within a molecule that is responsible for the characteristic chemical reactions of the molecule.
  • exemplary functional groups include, without limitation, alkyl, alkenyl, alkynyl, aryl, halo (fluoro, chloro, bromo, iodo), epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate ester, carboxylate, carboxyl, ether, ester, peroxy, hyrdoperoxy, carboxamide, amino (primary, secondary, tertiary), ammonium, imide, azide, cyanate, isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkyl, nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), disulfide.
  • halo generally refers to fluoro, chloro, bromo, or iodo.
  • heteroaryl generally refers to an aryl group comprising at least one heteroatom, which can be selected from, but not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorous, and oxidized forms thereof within the ring.
  • Heteroaryl groups can comprise a single ring or multiple condensed rings, wherein the condensed rings may or may not be aromatic and/or contain a heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl group.
  • Heteroaryl groups may be substituted with one or more groups other than hydrogen, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • a fluorophore can also be described herein as a heteroaryl group.
  • hydroxyl generally refers to the group —OH.
  • mine generally refers to an organic compound containing a —C ⁇ NR group, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • immunoglobin G generally refers to one of the primary classes of immunoglobins having heavy chains known as gamma-chains.
  • isoelectric point (pI) generally refers to the pH at which a molecule carries no net electric charge.
  • Polymeric molecules such as proteins comprised of amino acids, can be positive, neutral, negative, or polar in nature, giving the polymeric material an overall charge.
  • a molecule with a low pI value carries a net negative charge at neutral pH, and a molecule with a high pI value carries a net positive charge at a neutral pH.
  • MWCO molecular weight cut-off
  • multimodal generally refers to the ability of a material or compound, such as a resin or matrix, to provide plural different types of interactions between the resin or matrix and a desired molecule to contribute to the separation of a first desired molecule from a second molecule, such as by retention of the first and/or second molecule to the resin or matrix.
  • negatively charged small molecule generally refers to a molecule with a low isoelectric point (pI), which carries a net negative charge at a neutral pH.
  • phosphate generally refers to —O—P(O)(OR a ) 2 , wherein each R a independently is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other organic functional group.
  • positively charged large molecule generally refers to a molecule with a high isoelectric point (pI), which carries a net positive charge at a neutral pH.
  • sample generally refers to any fluid or solution comprising at least two molecules that need to be separated, wherein a first of the at least two molecules is a small molecule, as defined herein, and a second of the at least two molecules is a large molecule, as defined herein.
  • the sample may comprise a small molecule, such as but not limited to a stabilizing molecule.
  • the stabilizing molecule can be a purified protein isolated from natural or recombinant sources.
  • a recombinant albumin for example, a recombinant albumin, a native albumin, human serum albumin, an albumin-like stabilizer, bovine serum albumin, comparable mammalian serum (such as but not limited to, rabbit serum and mouse serum), ovalbumin, glycerol, and/or gelatin.
  • a sample can also include one or more molecules derived from biological samples.
  • the terms “separation,” “extraction,” “extracted,” “removal,” “reducing” or “reducing the quantity of,” or “purification” generally refer to removing or isolating a substance, e.g., a small molecule, such as BSA, or a large molecule or a biomolecule, such as IgG, from a mixture comprising the small molecule and/or large molecule.
  • a substance e.g., a small molecule, such as BSA, or a large molecule or a biomolecule, such as IgG
  • An extracted substance or a sample from which a substance has been extracted has significantly decreased quantities of components that were present in the sample prior to separation, and the extracted substance can be substantially reduced, substantially removed, substantially concentrated, substantially pure, or pure (devoid of any contaminants), compared to prior to being extracted.
  • small molecule and “large molecule” which refer to species to be separated from one another.
  • Small molecule or “smaller molecule” generally refers to any molecule having a molecular weight of less than 100 kDa
  • a “large molecule” is a molecule having a molecular weight of greater than or equal to 100 kDa.
  • the small molecule (such as but not limited to a biomolecule) may be being used to treat, derivatize, conjugate, cross-link, label, tag, chemically or biologically modify the large molecule for further analysis.
  • the small molecule can be a stabilizing molecule, such as but not limited to, a purified protein isolated from natural or recombinant sources.
  • a recombinant albumin for example, a recombinant albumin, a native albumin, human serum albumin, an albumin-like stabilizer, bovine serum albumin, comparable mammalian serum, ovalbumin and/or gelatin.
  • Derivatization includes labeling molecules with labels such as dyes, affinity tags, radioactive labels, mass tags, metals, and the like.
  • Derivatization also includes chemically modifying molecules by reduction, oxidization, methylation, biologically or biochemically modifying biomolecules, etc.
  • Biomolecules include, again without limitation: tagged proteins or nucleic acids; labeled biomolecules that are labeled with a variety of labels such as but not limited to dyes, fluorescent dyes, radioactive labels, affinity labels, mass-tags, metals, etc.; conjugated biomolecules, including conjugated antibodies; biomolecules conjugated to nanoparticles; metals, such as gold conjugated to nanoparticles; dyes or labels, such as biotin conjugated to toxins; chemical derivatives of biomolecules, such as but not limited to, reduced proteins, oxidized proteins, methylated nucleic acids, and proteins with sulfhydryl modified proteins.
  • Large molecules and/or biomolecules include, for example and without limitation, proteins, glycoproteins, and antibodies.
  • the small molecule is BSA having a molecular weight of 67,000 Da.
  • the large molecule is IgG having a molecular weight of 150 kDa.
  • a sample comprises a mixture of BSA and IgG.
  • size exclusion support generally refers to an inert porous solid that has a porosity which determines the size of a molecule that may be included or excluded from entering the pores.
  • the pores of a porous size exclusion support have a molecular size cut-off (MWCO) of 40 kDa or greater than 40 kDa.
  • sulfonamide generally refers to the group —SO 2 amiI or —N(R)sulfonyl, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • sulfonate generally refers to —SO 3 ⁇ , wherein the negative charge of the sulfonate group may be balanced with a positive counterion, such as an M + counter ion, wherein M + may be an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R b ) 4 , where R b is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality; or an alkaline earth ion, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0 0.5 .
  • M + may be an alkali ion, such as K + , Na +
  • R represents the rest of the molecule to which the sulfonyl group is bound and R′ is selected from hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • R represents the rest of the molecule to which the thioester group is bound and R′ is selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • thioether generally refers to a functional group with the general formula: R—S—R′ where R and R′ independently are selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • R and R′ independently are selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functionality.
  • a thioether is similar to an ether, except that a thioether contains a sulfur atom in place of the oxygen atom of an ether.
  • Certain disclosed embodiments of the present disclosure concern a matrix for separating molecules, such as biomolecules, from each other.
  • molecules are separated from each other using differences in one or more properties such as the size of the molecules, charge of the molecules, the isoelectric point (pI) of the molecules, and/or a combination of these properties.
  • molecules can be separated from each other based on one or more separation matrix properties such charge on the matrix and size exclusion properties of the matrix.
  • the matrix may comprise a porous size exclusion support, at least one cationic moiety, and may further be equilibrated with an equilibration buffer.
  • a sample solution comprising at least one small molecule and at least one large molecule is applied to the matrix, wherein the large molecule elutes faster than the small molecule that is trapped by the matrix.
  • Presently disclosed matrixes provide unexpectedly rapid, economical, and efficient separation of small molecules from large molecules.
  • the small molecule can be, but is not limited to, a protein, globular protein, sphero protein, serum albumin protein, polypeptide, and/or the like.
  • the one or more small molecules can be separated from one or more larger molecules using one or more properties, including, but not limited to, the isoelectric point of the molecules.
  • the one or more small molecules can be one or more negatively charged small molecules.
  • the one or more negatively charged small molecules can have an isoelectric point (pI) value in the range of 4.5-5.5.
  • the one or more negatively charged small molecules may have a pI value of 4.5, 4.6, 4.7 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, and/or 5.5.
  • the one or more negatively charged small molecule has a pI value of 4.9.
  • the matrix separates, extracts, removes, and/or reduces the quantity of one or more small molecules from one or more large molecules based on, but not limited to, the size of the molecules.
  • the one or more small molecule can have a molecular weight in the range of less than 100 kDa, less than 80 kDa, and/or less than 70 kDa.
  • the one or more small molecules may have a molecular weight of from 50 kDa to 80 kDa, such as 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kD, 58 kDa, 59 kDa, 60 kDa, 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, 69 kDa, 70 kDa, 71 kDa, 72 kDa, 73 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, 79 kDa, and/or 80 kDa.
  • One embodiment of the present disclosure describes matrixes for separating, extracting, removing, and/or reducing the quantity of one or more small molecules from one or more large molecules.
  • the one or more large molecules can be, but is not limited to, a glycoprotein, phosphoprotein, antibody, or immunoglobulin.
  • the one or more small molecules can be separated from one or more larger molecules using one or more properties including, but not limited to, the isoelectric point of the molecules.
  • the one or more large molecules can be one or more positively charged large molecules.
  • the one or more positively charged large molecules may have an isoelectric point (pI) value in the range of 8.0-11.5.
  • the one or more positively charged large molecules may have a pI value of 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, and/or 11.5.
  • the one or more positively charged large molecules has a pI value of 11.0.
  • the matrix separates, extracts, removes, and/or reduces the quantity of one or more small molecules from one or more large molecules based on, but not limited to, the size of the molecules.
  • the one or more large molecules may have a molecular weight greater than or equal to 100 kDa, and in certain embodiments a molecular weight greater than or equal to 150 kDa.
  • Matrixes according to the present disclosure may comprise a porous size exclusion support.
  • a porous size exclusion support may comprise spherical beads made of a gel or a gel-like material having pores.
  • the pore size range of a porous size exclusion support determines the size of a molecule that may be included or excluded from entering the size exclusion support. Without being bound by a single theory, it currently is believed that when a sample solution is passed through a size exclusion support, at least one small molecule in the sample enters the pores of the size exclusion support and is forced to follow a circuitous path before later exiting the size exclusion support. On the other hand, larger molecules take a relatively direct path through the size exclusion support.
  • exemplary size exclusion supports are made of agarose, polyacrylamide, cellulosic materials, and/or derivatives thereof.
  • a porous size exclusion support may comprise an agarose support, a polyacrylamide support, a cellulosic material support, or derivatives thereof.
  • a porous size exclusion support comprises HEC.
  • the porous size exclusion support is produced by using sufficient HEC to produce a resin with a MWCO greater than or equal to 40 kDa.
  • the porous size exclusion support is produced from a range of 50 grams (g) to 250 grams (g) HEC, such as, but not limited to 50 g HEC to 150 HEC g.
  • 80 g of HEC was used to produce the porous size exclusion support.
  • 90 g of HEC was used to produce the porous size exclusion support.
  • 108 g of HEC was used to produce the porous size exclusion support.
  • 129 g of HEC was used to produce the porous size exclusion support.
  • 147 g of HEC was used to produce the porous size exclusion support.
  • 216 g of HEC was used to produce the porous size exclusion support.
  • the porous size exclusion support is produced with 60 g HEC to 130 g HEC at a reaction scale of from greater than 0 liters (L) to 10 liters (L), such as, but not limited to, from greater than 0 L to 5 L, to produce the size exclusion support.
  • 0.5 L to 2 L of resin bed comprising the porous size exclusion support is produced from 60 g HEC to 130 g HEC.
  • 0.5 L to 1.5 L of resin bed comprising the porous size exclusion support is produced from 80 g HEC to 130 g HEC used in a 5 L reaction scale.
  • the size exclusion support can be crosslinked with a crosslinker such as, but not limited to, an epoxide-containing compound comprising at least one epoxide functional group, such as 1, 2, 3, or 4 epoxide groups.
  • the epoxide-containing compound may comprise one or more halo functional groups, aliphatic functional groups, heteroaliphatic functional groups, or a combination thereof.
  • the heteroaliphatic functional group can comprise a polyethylene glycol (or “PEG”) spacer arm.
  • the epoxide-containing compound can have Formula I,
  • R is a C 1 -C 10 alkyl
  • LG can be a halo functional group or a glycidol moiety.
  • the epoxide-containing compound can be epichlorohydrin (Epi), having a structure of
  • the epoxide-containing compound can be 1,4-butanediglycidylether.
  • the porous size exclusion support can be crosslinked with a range of 250 milliliters (mL) to 700 milliliters (mL) of the crosslinker
  • 265 mL of Epi was used to crosslink the porous size exclusion support.
  • 303 mL of Epi was used to crosslink the porous size exclusion support.
  • 375 mL of Epi was used to crosslink the porous size exclusion support.
  • 397 mL of Epi was used to crosslink the porous size exclusion support.
  • 410 mL of Epi was used to crosslink the porous size exclusion support.
  • 441 mL of Epi was used to crosslink the porous size exclusion support.
  • 662 mL of Epi was used to crosslink the porous size exclusion support.
  • the porous size exclusion support can be crosslinked by providing 250 mL Epi to 700 mL Epi at a reaction scale from greater than 0 L to 5 L. In some particular aspects of the present disclosure, greater than 0 L to 2 L of resin bed comprising the porous size exclusion support can be produced from 250 mL Epi to 400 mL Epi 5 L reaction scale.
  • the porous size exclusion support has a MWCO greater than or equal to 40 kDa and hence molecules that are excluded have a molecular weight of greater than or equal to 40 kDa.
  • the pore size of the porous size exclusion support may have a MWCO size for excluding from the pores molecules of greater than or equal to 40 kDa to 150 kDa, more typically from 50 kDa to 150 kDa, and even more typically from greater than or equal to 40 kDa to 60 kDa, including a MWCO in between, such as but not limited to, greater than or equal to 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 k
  • the porous size exclusion support has a MWCO of greater than or equal to 40 kDa. In another exemplary embodiment, the porous size exclusion support has a MWCO of 45 kDa. In another exemplary embodiment, the porous size exclusion support has a MWCO of 50 kDa. In another exemplary embodiment, the porous size exclusion support has a MWCO of 80 kDa. In another exemplary embodiment, the porous size exclusion support has a MWCO of 90 kDa.
  • Matrixes according to the present disclosure may comprise a porous size exclusion support and a cationic moiety.
  • small molecules such as, but not limited to, negatively charged molecules can be separated from large molecules, such as, but not limited to, positively charged molecules based on one or more matrix properties.
  • the one or more matrix properties can be the charge and/or the size exclusion properties of the matrix.
  • the cationic moieties of a matrix of the disclosure are amines, imines, or a combination thereof.
  • such cationic moieties may comprise an amine-containing polymer; alkylamines, particularly lower alkyl amines (e.g., pentylamines); amine-containing heterocyclic compounds; amine-containing aromatic compounds; or other amine/diamine compounds.
  • the amine-containing polymer prior to association with the porous size exclusion support, is branched polyethylene imine (PEI), having a base structure as shown below, which when exposed to suitable conditions, such as but not limited to, an equilibration buffer, becomes charged to provide the cationic moiety.
  • PEI polyethylene imine
  • the amine-containing polymer prior to association with the porous size exclusion support, is a diamine comprising one or more polyethylene glycol groups, such as 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine (also known commercially as Jeffamine) having a base structure as shown below.
  • suitable conditions such as but not limited to, an equilibration buffer, the diamine becomes charged to provide the cationic moiety.
  • the alkyl diamine prior to association with the porous size exclusion support, is diaminopentane (PDA), having a structure NH 2 (CH 2 ) 5 NH 2 (and also shown below), which when exposed to suitable conditions, such as but not limited to, an equilibration buffer, becomes charged to provide the cationic moiety.
  • PDA diaminopentane
  • the diamine prior to association with the porous size exclusion support, is N,N diethylethylenediamine (DEED), having a structure as shown below, which when exposed to suitable conditions, such as but not limited to, an equilibration buffer, becomes charged to provide the cationic moiety.
  • DEED N,N diethylethylenediamine
  • the amine-containing aromatic compound may comprise at least one aromatic ring having from C 3 to C 15 and at least one amine-containing compound.
  • the amine containing-compound can have general Formula II, or an enantiomer, a diastereomer, a tautomer, a salt, a solvate and/or an isotopically substituted derivative thereof
  • J, Q, T, X, Y, and Z are the same or different, and each of J, Q, T, X, Y, and Z independently is selected from nitrogen or CR c , wherein R c , for each occurrence, independently is selected from hydrogen, halo, aliphatic, heteroaliphatic, or amino.
  • each RC can be the same or different.
  • each of J, Q, T, X, Y, and Z are CR c and at least two R c groups comprise an amino group and the remaining RC groups are hydrogen.
  • a compound of Formula II can be selected from, but not limited to, 1,2-diaminobenzene having a structure shown below, 1,3-diaminobenzene having a structure shown below, and/or 1,4-diaminobenzene having a structure shown below.
  • 1,2-diaminobenzene having a structure shown below 1,3-diaminobenzene having a structure shown below
  • 1,4-diaminobenzene having a structure shown below.
  • an amine-containing heterocyclic compound can be associated with the size exclusion support comprising HEC.
  • an epoxy amine compound can be associated with the size exclusion support comprising HEC.
  • the amine-containing can be an epoxy amine, such as, but not limited to, (S)-N-boc-2,3-epoxypropylamine having a base structure below, which when exposed to suitable conditions, such as but not limited to, an equilibration buffer, becomes charged to provide the cationic moiety.
  • a matrix of the disclosure comprises at least one cationic moiety that is associated with a porous size exclusion support.
  • a porous size exclusion support or a cationic moiety can include a reactive functional group.
  • a functional group on a porous size exclusion support can be used to interact with and associate with one or more cationic moieties to form a matrix.
  • a matrix comprises at least one cationic moiety that is immobilized onto at least one size exclusion support.
  • the cationic moiety may be “immobilized” by being covalently bound to the size exclusion support by formation of a covalent bond.
  • the covalent bond can be formed using alkylation or by forming an amide or amine bond between a porous size exclusion support and a cationic moiety.
  • immobilization according to the disclosure is achieved by first oxidizing hydroxyl groups of HEC to aldehyde groups using any suitable oxidizing agent, with some examples using a periodate (IO 4 ⁇ or IO 6 5 ⁇ ).
  • the aldehyde groups generated by oxidation can react with terminal amines on a cationic moiety to covalently bind the cationic moiety to the HEC support, and the resulting intermediate may be reduced by using a reducing agent, such as, but not limited to, sodium cyanoborohydride or picoline borane, to form an amine.
  • a reducing agent such as, but not limited to, sodium cyanoborohydride or picoline borane
  • the epoxy amine can be immobilized onto the size exclusion support.
  • the epoxy amine can associate with and/or immobilize onto the size exclusion support comprising HEC by reacting with a hydroxyl group provided by the HEC.
  • the epoxy amine compound is a protected amine compound, wherein the protected amine compound is deprotected after associating with or immobilizing onto the size exclusion support.
  • the cationic moiety associates with the one or more small molecules, such as, but not limited to, a negatively charged small molecule.
  • the negatively charged small molecule can associate with the cationic moiety through interactions and/or bonds, such as, but not limited to, an ionic interaction, a hydrophilic interaction, a hydrophobic interaction, an affinity interaction, hydrogen bonding, Van der Waals forces, and/or covalent bonding.
  • a functional group on a cationic moiety can react with a functional group on a porous size exclusion support and/or a small molecule, such as but not limited to, a negatively charged small molecule, to be separated or extracted from the at least one large molecule.
  • Functional groups can include, but are not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, and/or any other functional group suitable for associating and/or interacting cationic moieties with the porous size exclusion support and/or small molecule.
  • functional groups include at least one reactive group represented by either R x , which represents a reactive functional moiety; or (—L—R x ), which represents a reactive functional moiety R x that is attached to either a porous size exclusion support or a moiety by a covalent linkage L.
  • the reactive group functions as the site of association, attachment and/or interaction with a moiety or a small molecule wherein the reactive group chemically reacts with an appropriate reactive or functional group on the porous size exclusion support, the moiety, or the small molecule.
  • a reactive group or a functional group can be an acrylamide, an activated ester of a carboxylic acid, an acyl halide group, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a thioboronate group, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, a thiol group, a sulfide group, a disulfide group, an epoxide group, and episulfide
  • a reactive group or functional group can comprise electrophiles and/or nucleophiles and can, in some embodiments, generate a covalent linkage between them.
  • Exemplary electrophiles and nucleophile functional groups can an aryloxy group or aryloxy substituted one or more times by electron-withdrawing substituents such as nitro, fluoro, chloro, cyano, trifluoromethyl, or combinations thereof, used to form activated aryl esters; or a carboxylic acid activated by a carbodiimide to form an anhydride or mixed anhydride —OCOR a or —OCNR a NHR b , where R a and R b , which may be the same or different, are C 1 -C 6 alkyl, C r C 6 perfluoroalkyl, or C 1 -C 6 alkoxy; or cyclohexyl, 3-dimethylaminopropyl, an acyl halide group, an acyl
  • the reactive group further comprises a linker, L, in addition to the reactive functional moiety.
  • the linker can be used to covalently attach a reactive functional group.
  • the linker is a single covalent bond or a series of stable bonds.
  • a reactive functional moiety may be directly attached (where the linker is a single bond) through a series of stable bonds, to the solid support, the moiety or to the small molecule.
  • the linker typically incorporates several nonhydrogen atoms selected from C, N, O, S, Si, B and P.
  • the covalent linkage can incorporate a platinum atom, such as described in U.S. Pat. No. 5,714,327.
  • the linker When the linker is not a single covalent bond, the linker may be any combination of stable chemical bonds, optionally including, single, double, triple, or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds.
  • the linker incorporates less than 15 nonhydrogen atoms and is composed of a combination of ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazide, aromatic, and/or heteroaromatic bonds.
  • the linker is a single covalent bond or a combination of single carbon-carbon bonds and carboxamide, sulfonamide, or thioether bonds.
  • the following moieties can be found in the linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and amine moieties.
  • L include substituted or unsubstituted polymethylene, arylene, alkylarylene, aryl, or arylthiol.
  • linkers can be used to attach functional or reactive groups.
  • the reactive group is a maleimide or haloacetamide
  • the resulting compound is particularly useful for conjugation to thiol-containing substances.
  • the reactive group is a hydrazide
  • the resulting compound is particularly useful for conjugation to periodate-oxidized carbohydrates and glycoproteins.
  • the cationic moiety may comprise an open and/or unreacted amine.
  • a matrix comprising a size exclusion support and a cationic moiety, such as, but not limited to, PDA may comprise an open/unreacted amine.
  • the open/unreacted amine on PDA can be reacted with a polysaccharide derived molecule having a molecular weight range from 0 to 2,000,000.
  • the open and/or unreacted amine on the cationic moiety PDA is reacted with dextran having a molecular weight of 1,000,000.
  • the concentration of the cationic moiety that is added to the matrix can be in the range of from 50 mg/mL to 175 mg/mL. In some embodiments the concentration of the cationic moiety is from 50 mg/mL to 100 mg/mL, from 50 mg/mL to 75 mg/mL, from 50 mg/mL to 150 mg/mL, from 100 mg/mL to 155 mg/mL, from 130 mg/mL to 175 mg/mL. In one exemplary embodiment, the concentration of the cationic moiety can be 50 mg/mL. In another exemplary embodiment, the concentration of the cationic moiety can be 75 mg/mL. In another exemplary embodiment, the concentration of the cationic moiety can be 150 mg/mL.
  • FIG. 1 depicts a non-limiting exemplary embodiment, where a porous size exclusion support 10 is produced with 129 grams of HEC and 265 milliliters of Epi to make 1.5 liters of resin bed, and wherein the HEC is modified with a cationic PDA moiety 20 to form matrix 30.
  • Matrix 30 has a MWCO of 50 kDa, and an overall positive charge.
  • FIG. 2 depicts another non-limiting exemplary embodiment of a porous size exclusion support 10 that is produced with 129 grams of HEC and 265 milliliters of Epi to make 1.5 liters of resin bed, and wherein the HEC is modified with a branched PEI cationic moiety 40 to form matrix 50.
  • Matrix 50 has a MWCO of 50 kDa, and an overall positive charge.
  • FIG. 3 depicts another non-limiting exemplary embodiment of a porous size exclusion support 10 is produced with 129 grams of HEC and 265 milliliters of Epi to make 1.5 liters of resin bed, and wherein the HEC is modified with a DEED cationic moiety 60 to form matrix 70.
  • Matrix 70 having a MWCO of 50 kDa, and an overall positive charge.
  • a buffer can be provided such that it increases the binding capacity of the matrix to smaller sample components while also decreasing the binding capacity of the matrix to larger sample components.
  • a buffer increases the binding capacity of the cationic moiety to a small molecule in a sample comprising the small molecule, such as but not limited to, a negatively charged small molecule; and decreases the binding capacity of the cationic moiety to a large molecule, such as, but not limited to a positively charged large molecule.
  • an additional buffer can be used to improve the recovery of larger sample components.
  • the buffer is an equilibration buffer.
  • the equilibration buffer contains a substantially free of, or is free of, a salt, such as, but not limited to, NaCl.
  • An equilibration buffer comprising a substantially free amount of salt generally refers to greater than 0 mM of salt to 10 mM of salt, preferably from greater than 0 mM of salt to 5 mM of salt.
  • the equilibration buffer can have a pH of 4 to 9
  • the equilibration buffer can have a positive charge.
  • the equilibration may comprise a neutral charge.
  • the equilibration buffer having a positive charge can include, but is not limited to, carbonate buffers, bicarbonate buffers, phosphate buffers, citric acid/citrate buffers, and combinations thereof.
  • the buffer may comprise a Tris buffer, that is a buffer solution comprising 2-amino-2-(hydroxymethyl)propane-1,3-diol, also referred to as tris(hydroxymethyl)aminomethane; and triethylammonium bicarbonate.
  • the equilibration buffer can have a pH of 4 to 9, such as from pH of 5 to 8, such as from pH of 5 to 7.
  • the equilibration buffer has a pH of 5, pH of 7, and/or pH of 8.5.
  • the equilibration buffer is sodium acetate having a pH of 5.
  • the equilibration buffer is sodium acetate having a pH of 7.
  • the equilibration buffer is HEPES having a pH of 5.
  • the equilibration buffer is HEPES having a pH of 7.
  • the equilibration buffer is borate having a pH of 5.
  • the equilibration buffer is borate having a pH of 8.5.
  • the equilibration buffer is a charged buffer with no salt (e.g., no sodium chloride).
  • the equilibration buffer is used at a concentration from 1 mM to 100 mM, such as from 10 mM to 100 mM, from 20 mM to 100 mM, from 40 mM to 100 mM, or from 50 to 100 mM. In an exemplary embodiment, the equilibration buffer concentration is 50 mM.
  • FIG. 4 illustrates a process for separating a small molecule 90 from a sample comprising a large molecule 100 using a matrix 80 having a cationic moiety 82 associated therewith.
  • Small molecule 90 associates with the cationic moiety 82 to form composition 110 .
  • a positively charged buffer can be used to facilitate the separation of a small molecule (e.g., a negatively charged molecule, such as BSA), from a large molecule (e.g., a positively charged molecule, such as IgG).
  • the present disclosure also concerns embodiments of a system comprising one or more disclosed matrixes and further comprising a container.
  • Such systems provide one or more advantages, including, but not limited to: economical feasibility; simplicity and ease of use; providing faster results relative to conventional products in the art; adaptability as a single use disposable unit; adaptability for high throughput sample preparation in multi-well container formats; and adaptability for automated and robotic sample preparation systems. Reducing the concentration of small molecules in a sample using the systems provided here provides quick recovery of biomolecules, as well as superior purity of biomolecules and their derivatives that can be used for downstream applications.
  • the present disclosure provides a system for removing one or more small molecules from a sample using differences in one or more properties, such as, but not limited to, the size of the molecules, charge of the molecule, isoelectric point (pI) of the molecules, and/or any combination of these properties.
  • the system can comprise: (i) a container having at least one size exclusion support and at least one cationic moiety that can associate with the one or more small molecules; and (ii) a receptacle located to receive flow from the container. Additionally, molecules can be separated from each other based on one or more separation matrix properties, such as the charge and size exclusion properties of the size exclusion support associated with the at least one cationic moiety.
  • the receptacle is attached to the column. In some embodiments, the receptacle is detachable from the column. The contents of a receptacle can be used or removed by a user as a desired. In some embodiments, the receptacle collects sample with substantially reduced small molecules. In some embodiments, the receptacle collects sample with no small molecules.
  • the system may be operably configured to operate by gravity flow.
  • an external, affirmatively-applied pressure or force can be applied to facilitate flow, such as a centrifugal force, a positive pressure, a negative pressure, vacuum and combinations thereof.
  • Structures that allow applications of the above-mentioned pressures or forces include, without limitation: a syringe that can be drawn to cause a positive pressure; a vacuum frit for generating negative pressure; and/or tubes or containers adaptable to commercially available centrifuges or rotatory devices.
  • the system can be configured for use with or in a centrifuge tube or any other comparable rotary instrument.
  • the container is a columnar container, a tube, a multi-well tube, a multi-well plate or a multi-well filter plate.
  • Exemplary containers include, but are not limited to, a test tube, a spin column, a multi-well plate, a multi-well filter plate, a micro-well plate, or a micro-well filter plate.
  • FIG. 5 depicts an exemplary separation system 200 comprising a container 240 and a sample flowing through the container.
  • the sample comprises a small molecule 210 and a large molecule 230 .
  • Container 240 houses matrix 220 that is configured to separate the small molecule 210 from the large molecule 230 using differences in one or more properties such as but not limited to, the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and/or combination of these properties.
  • System 200 also includes a receptacle 250 for recovering the large molecule 230 .
  • FIG. 6 depicts an exemplary separation system 300 according to one embodiment of the present disclosure.
  • System 300 comprises: a container 310 (such as a columnar tube, a test-tube or a spin column); a matrix 320 housed by the container 310 ; a receptacle 330 located below the container 310 that is adaptable or configured to receive fluid flowing through the container 310 through its bottom end 340 .
  • container 310 may also comprise one or more frits (not depicted).
  • system 300 can include an optional lid 350 that can be used to secure container 310 at a top end 360 .
  • receptacle 330 can be detached from container 310 so that a user can collect the flow-through.
  • receptacle 330 has a twist-off tab configuration for removal.
  • a receptacle 330 can be threadedly coupled to container 310 , or attached using a complementary fit that can be pulled apart, and the like.
  • FIG. 7 is a perspective view of an exemplary system 400 according to one embodiment of the present disclosure comprising a multi-well container 410 .
  • Multi-well container 410 comprises a wall that defines multiple wells 412 .
  • the wall may comprise an end 420 , a side 430 , and a top 440 that defines multiple wells.
  • Multi-well container 410 houses a matrix comprising at least one size exclusion support and at least one cationic moiety according to the present disclosure that can associate with the one or more small molecules using the properties, such as, but not limited to, the size, the charge, the isoelectric point (pI), and/or any combination of these properties.
  • the multi-well container may comprise an optional container lid 450 .
  • Lid 450 can be any suitable removable/detachable structure, such as a foil, a clear wrap, or a tear-off seal.
  • Multi-well container 410 can be a multi-well plate, a multi-well plate filter, a microplate or a microtiter plate comprising a flat plate comprising multiple-wells 412 where each well is used as a small test tube or container.
  • Multi-well plates come in a variety of formats for high-throughput use and may comprise 6, 12, 24, 48, 96, 384, 1536, 3456, 9600 or more wells arranged in a rectangular matrix or array.
  • FIG. 8 depicts an exemplary system 500 according to one embodiment comprising a multi-well container 510 .
  • System 500 further comprises a receptacle 550 located below the container 510 that is adaptable or configured to receive fluid flowing from the container.
  • Container 510 comprises an end 520 , a top 540 , and a side 530 .
  • receptacle 550 may comprise a multi-well tray to collect flow-through.
  • Receptacle 550 may be detachable and can be collected by a user.
  • receptacle 550 is a wash plate or a collection plate.
  • Systems according to the present disclosure may be fully automated or may be manually operated systems.
  • a system may be operated in part manually and in-part by automation.
  • a system can also comprise a computer system comprising a CPU, hardware elements, and/or software elements. Suitable computer systems may be operable to control various components of the system, such as a control robot to retrieve flow-through and analyze flow-through. In some embodiments, a computer system and/or components thereof may reside physically within system 300 , 400 , or 500 , or may reside externally.
  • a computer system used herein may comprise a data analysis and control system, a data transfer system such as a read-write CD ROM Drive or DVD drive, at least one USB port, and/or at least one Ethernet port.
  • a computer system may include pre-loaded software and/or Application Specific Integrated Circuits (ASICS) to control disclosed systems, such as systems 300 , 400 , 500 , and/or other components the system, including sample processing and analysis, display, and/or exporting the results.
  • ASICS Application Specific Integrated Circuits
  • Disclosed system embodiment also may optionally comprise one or more devices or components operable to further process the flow-through.
  • the flow-through can be eluted molecules, such as but not limited to, eluted larger sample components, such as one or more large molecules.
  • a system may also comprise additional devices or components, such as but not limited to, a power supply, a display unit, such as a monitor operable to view sample processing and/or to monitor extraction of biomolecules from samples; spectrophotometers; devices to measure nucleic acid extraction; devices to further process extracted biomolecules for further analysis; printers and the like.
  • a system of the disclosure may be configured to fit on a laboratory bench top.
  • Embodiments of the present disclosure also concern a method for making a matrix or system according to the present disclosure.
  • the method of making comprises immobilizing a cationic moiety to a porous size exclusion support.
  • a porous size exclusion support may comprise spherical beads made of a gel or a gel-like material having pores.
  • Some exemplary size exclusion supports comprise agarose, polyacrylamide, cellulosic materials (e.g., hydroxyethyl cellulose), and/or derivatives thereof.
  • the porous size exclusion support may be crosslinked with at least one crosslinker.
  • the pore size range of a porous size exclusion support determines the size of a molecule that may be included or excluded from entering the porous size exclusion support. Without being bound by this theory, it currently is believed that when a sample solution is passed through a porous size exclusion support molecules having a molecular weight less than or substantially equal to the MWCO are forced to follow a circuitous path before later exiting the porous size exclusion support. On the other hand, large molecules take a relatively direct path through the porous size exclusion support. Therefore, the difference in flow rates between the small molecules and large molecules allows for separating the faster-flowing large molecules from the slower-flowing small molecules as a sample travel through the size exclusion support.
  • Disclosed embodiments of the porous size exclusion support may comprise products formed by reacting 50 grams (g) to 250 grams (g) HEC.
  • disclosed size exclusion supports have may comprise HEC ranging from 80 grams to 130 grams HEC for a 1.5 L resin bed.
  • 80 g of HEC was used to produce the porous size exclusion support.
  • 90 g of HEC was used to produce the porous size exclusion support.
  • 108 g of HEC was used to produce the porous size exclusion support.
  • 129 g of HEC was used to produce the porous size exclusion support.
  • 147 g of HEC was used to produce the porous size exclusion support.
  • 216 g of HEC was used to produce the porous size exclusion support.
  • the porous size exclusion support is produced by crosslinking HEC with a crosslinker.
  • HEC can be crosslinked with an epoxide, such as but not limited to, epichlorohydrin (Epi).
  • Epi epichlorohydrin
  • the porous size exclusion support can be crosslinked with a range of 250 mL to 450 mL Epi.
  • the support can be produced from 250 mL to 450 mL of epichlorohydrin for a 1.5 L resin bed.
  • 265 mL of Epi was used to crosslink the porous size exclusion support.
  • 303 mL of Epi was used to crosslink the porous size exclusion support.
  • 375 mi., of Epi was used to crosslink the porous size exclusion support.
  • 397 mL of Epi was used to crosslink the porous size exclusion support.
  • 410 mL of Epi was used to crosslink the porous size exclusion support.
  • 441 mL of Epi was used to crosslink the porous size exclusion support.
  • the cationic moiety is immobilized to the porous exclusion support by formation of a covalent bond.
  • the covalent bond can be formed by reactions, such as, but not limited to, amidation, alkylation, amination, or other covalent bond-forming reactions.
  • a functional group of the size exclusion support e.g., hydroxyl group
  • oxidized such as by using a periodate, to generate an aldehyde.
  • 15-35 mg/mL of the oxidation agent such as a periodate
  • the oxidation agent such as a periodate
  • 20 mg/mL 21 mg/mL, 22 mg/mL 23 mg/mL, 24 mg/mL 25 mg/mL, 26 mg/mL, 27 mg/mL, 28 mg/mL, 29 mg/mL, 30 mg/mL, 31 mg/mL, 32 mg/mL, 33 mg/mL, 34 mg/mL, 35 mg/mL, of a periodate is added to oxidize the size exclusion support.
  • the size exclusion support is oxidized using sodium periodate.
  • 5-15 mg/mil of the reducing agent is added. In some embodiments, 5 mg/ mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, of the reducing agent is added.
  • the reducing agent is sodium cyanoborohydride. In another exemplary embodiment, the reducing agent is picoline borane.
  • the cationic moieties associated with a size support may comprise an amine group.
  • the cationic moiety comprises an amine-containing polymer, pentylamine groups, diamine, or an imine group.
  • the amine-containing polymer is branched PEI.
  • the diamine is PDA.
  • the diamine is DEED.
  • the concentration of the cationic moiety that is used to modify the support material can be in the range of 50 mg/mL to 175 mg/mL.
  • the concentration of the cationic moiety may comprise from 50 mg/mL to 170 mg/mL, from 60 mg/mL to 165 mg/mL, from 65 mg/mL to 160 mg/mL, from 70 mg/mL to 155 mg/mL, from 75 mg/mL to 150 mg/mL, from 80 mg/mL to 145 mg/mL, from 85 mg/mL to 140 mg/mL, from 90 mg/mL to 135 mg/mL, or from 95 mg/mL to 130 mg/mL.
  • the concentration of the cationic moiety is 50 mg/mL. In another exemplary embodiment, the concentration of the cationic moiety 75 mg/mL. In yet another exemplary embodiment, the concentration of the cationic moiety can be 150 mg/mL.
  • sodium metaperiodate is dissolved in water and is mixed with the porous size exclusion support matrix bed comprising ITEC to oxidize vicinal diols present in the HEC to aldehyde groups.
  • the mixture is allowed to react for a suitable time period, such as from 2 to 4 hours, at a temperature ranging from 15° C. to 30° C.
  • the mixture is allowed to react for at least four hours at room temperature with constant overhead stirring.
  • the cationic moiety is prepared at a pH from 8.0 to 8.5 and is added to the slurry.
  • a suitable reducing agent such as sodium cyanoborohydride, is added to the mixture, and the reaction is allowed to proceed for a suitable time period, such as from 8 to 12 hours at 20° C. 30° C. with stirring, and is washed with water and NaCl.
  • Certain disclosed embodiments concern a method for separating at least one large molecule from at least one small molecule using differences in one or more properties, such as, but not limited to, size of the molecule, charge of the molecules, the isoelectric point (pI) of the molecules, and/or any combination of these properties.
  • the method may comprise: applying a sample to a porous size exclusion support comprising at least one cationic moiety that can bind to or associate with a small molecule; and subjecting the container to a gravity flow, a centrifugal force, a positive pressure, a negative pressure, a vacuum or a combination thereof.
  • the large molecule in the sample can be excluded by the porous size exclusion support and is collected as flow-through.
  • the one small molecule can interact with the cationic moiety and is thereby separated from the large molecule in a sample using differences in one or more properties such as but not limited to the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and/or any combination of these properties.
  • properties such as but not limited to the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and/or any combination of these properties.
  • an equilibration buffer disclosed herein can be provided such that it increases the binding capacity of the matrix to smaller sample components while also decreasing the binding capacity of the matrix to larger sample components.
  • flow-through is collected in a receptacle located below the container.
  • small molecules may constitute a sample impurity or contaminant.
  • Samples that may be processed by methods of the disclosure may be any type of biological or clinical sample comprising biomolecules or derivatives thereof from which small molecules have to be separated or removed.
  • Some exemplary non-limiting samples include samples having the small molecule BSA and the large molecule IgG.
  • Methods of the disclosure advantageously reduce the time required for processing a sample, and/or increase the quantity of small molecules removed from the sample.
  • the present disclosure describes a kit for separating a large molecule from a small molecule using differences in one or more properties, such as, but not limited to, the size of the molecules, charge of the molecules, the isoelectric point of the molecules, and/or any combination of these properties.
  • the kit may comprise: a system comprising (i) a container housing a size exclusion support comprising a cationic moiety associated with the support, wherein the cationic moiety also can associate with a small molecule; and (ii) a receptacle located below the container, wherein the device is operably configured for gravity flow, or can operate by applying a centrifugal force, a positive pressure, a negative pressure, a vacuum, and combinations thereof.
  • kits of the disclosure are a spin column, a multi-well filter plate, or a multi-well plate.
  • a kit can further comprise one or more equilibration buffers packaged in one or more separate containers or included in the first container.
  • the equilibration buffer does not comprise a salt (e.g., an ionic salt like NaCl). In other aspects disclosed herein, the equilibration buffer may comprise a low amount of salt. In some aspects, the equilibration buffer can have a pH of 4 to 9 In another embodiment, the equilibration buffer can have a positive charge. In particular disclosed embodiments, the equilibration may comprise a neutral charge.
  • a salt e.g., an ionic salt like NaCl
  • the equilibration buffer may comprise a low amount of salt.
  • the equilibration buffer can have a pH of 4 to 9
  • the equilibration buffer can have a positive charge. In particular disclosed embodiments, the equilibration may comprise a neutral charge.
  • the equilibration buffer can include but is not limited to, carbonate buffers, bicarbonate buffers, phosphate buffers, citric acid/citrate buffers.
  • the buffer comprises a Tris Buffer.
  • the buffer comprises a TEAB buffer.
  • the equilibration buffer can have a pH of 4-9, such as from pH of 5 to 8, such as from pH of 5 to 7.
  • the equilibration buffer has a pH of 5, pH of 7, and/or pH of 8.5.
  • the equilibration buffer is sodium acetate having a pH of 5.
  • the equilibration buffer is sodium acetate having a pH of 7.
  • the equilibration buffer is HEPES having a pH of 5.
  • the equilibration buffer is HEPES having a pH of 7.
  • the equilibration buffer is borate having a pH of 5.
  • the equilibration buffer is borate having a pH of 8.5.
  • the positively charged equilibration buffer is present in a concentration from 1 mM to 100 mM, from 2 mM to 75 mM, from 5 mM to 50 mM, from 10 mM to 25 mM, from 5 to 25 mM. In some embodiments, the positively charged equilibration buffer is present in a concentration from 1 mM to 100 mM, from 2 mM to 75 mM, from 5 mM to 50 mM, from 10 mM to 25 mM, from 5 to 25 mM Tris Buffer. In an exemplary embodiment, the equilibration buffer concentration may be 50 mM.
  • a kit of the disclosure may also comprise one or more reagents such as one or more wash buffers, elution buffers, filter membranes and/or additional spin columns or multi-well plates.
  • one or more reagents such as one or more wash buffers, elution buffers, filter membranes and/or additional spin columns or multi-well plates.
  • kits may be comprised in one or more suitable containers.
  • a container may generally comprise at least one vial, test tube, flask, bottle, syringe or other container, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in a kit they may be packaged together if suitable or the kit will generally contain a second, third or other additional container into which the additional components may be separately placed. However, in some embodiments, certain combinations of components may be packaged together comprised in one container means.
  • a kit can also include a component for containing any reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • a component of a kit of the disclosure may be pre-filled with one or more of the reagents to process a sample and may be suitably aliquoted into appropriate chambers.
  • a kit or containers thereof may have a seal to keep the internal compartments and any contents therein sterile and spill proof.
  • kits Some components of a kit are provided in one and/or more liquid solutions.
  • Liquid solution may be non-aqueous solution, an aqueous solution, and may be a sterile solution.
  • Components of the kit may also be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that a suitable solvent may also be provided in another container means.
  • Kits may also comprise a container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits of the disclosure may also include instructions for employing the kit components and may also have instructions for the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.
  • a matrix comprising: a porous size exclusion support; and at least one cationic moiety associated with the porous size exclusion support, wherein the matrix can separate one or more molecules in a sample by using a molecular weight of the one more molecules, a charge of the one or more molecules, an isoelectric point (pI) of the one or more molecules, or a combination thereof.
  • pI isoelectric point
  • the porous size exclusion support has a molecular weight cut-off of greater than or equal to 40 kDa.
  • the at least one cationic moiety is covalently bound to the porous size exclusion support.
  • the at least one cationic moiety comprises an amine, a diamine, a polyamine, or an amine-containing polymer.
  • the amine-containing polymer is polyethylene imine.
  • the diamine is diaminopentane, N,N-diethylethylenediamine, or combination thereof.
  • the porous size exclusion support comprises hydroxyethyl cellulose.
  • the system configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or combinations thereof.
  • the container is a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.
  • Also disclosed herein is a method of making a multimodal resin, comprising: providing a porous size exclusion support comprising hydroxyethyl cellulose having a MWCO of greater than or equal to 40 kDa, the hydroxyethyl cellulose having at least one vicinal diol; oxidizing the least one vicinal diol to an aldehyde; and reacting the aldehyde with an amine group of a cationic moiety via reductive amination.
  • the porous size exclusion support is produced using an amount of hydroxyethyl cellulose ranging from 50 g to 250 g.
  • the method further comprising providing a crosslinker to crosslink the porous size exclusion support.
  • the crosslinker is epichlorohydrin.
  • the epichlorohydrin is used in an amount ranging from 250 mL to 450 mL.
  • the cationic moiety is diaminopentane, polyethylene imine, or N,N-diethylethylenediamine.
  • the cationic moiety has a concentration range from 50 mg/mL to 175 mg/mL.
  • Also disclosed herein is a method for separating one or more molecules in a sample, comprising: providing a matrix comprising a porous size exclusion support having at least one cationic moiety associated therewith, wherein the matrix can separate one or more molecules using a molecular weight of the one or more molecules, a charge of the one or more molecules, an isoelectric point (pI) of the one or more molecules, or a combination thereof; equilibrating the matrix with an equilibration buffer; and applying the sample to the matrix to separate the one or more molecules by subjecting the matrix to gravity flow, a centrifugal force, a positive pressure, a negative pressure, a vacuum, or a combination thereof, wherein the one or more molecules in the sample is excluded by the matrix and is collected as a flow-through, and wherein one or more molecules associates with the at least one cationic moiety and is thereby separated from the sample in a single step.
  • pI isoelectric point
  • the flow-through is collected in a receptacle located below the container.
  • the equilibration buffer is a positively charged buffer, a neutrally charged buffer, a low-salt buffer, or a salt-free buffer.
  • the equilibration buffer is sodium acetate having a pH of 5 or a pH of 7, HEPES having a pH of 5 or a pH of 7, or borate having a pH of 5 or a pH of 8.5.
  • the positively charged buffer is Tris Buffer or triethylammonium bicarbonate.
  • the first of the two or more molecules comprises: at least one small molecule having a molecular weight range of less than 100 kDa; and the second of the one or more molecules comprises least one large molecule having a molecular weight range of greater than or equal to 100 kDa.
  • the one or more molecules is bovine serum albumin.
  • the one or more molecules is an antibody.
  • the antibody is IgG.
  • the one or more molecules having an isoelectric point value range from 4.5 to 5.5 are separated from one or more molecules having an isoelectric point value from 8.0 to 11.5.
  • the one or more molecules are more negatively charged molecule from the other having positively charged molecules.
  • the at least one cationic moiety associates with the one or more molecules by ionic interaction, hydrophilic interactions, hydrophobic interactions, affinity interaction, hydrogen bonding, or van der Waals forces.
  • the one or more molecules comprises at least one negatively charged small molecule and at least one positively charged large molecule
  • the size exclusion support comprises HEC and Epi
  • the cationic moiety is diaminopentane; wherein greater than 80% of the at least one negatively charged molecule is separated and greater than 80% of the of the at positively charged large molecule is recovered as flow-through.
  • the one or more molecules comprises at least one negatively charged small molecule and at least one positively charged large molecule
  • size exclusion support comprises HEC and Epi
  • cationic moiety is diaminopentane
  • the cationic moiety is modified with dextran; wherein greater than 80% of the at least one negatively charged molecule is separated and greater than 80% of the of the at positively charged large molecule is recovered as flow-through.
  • the one or more molecules comprises at least one negatively charged small molecule and at least one positively charged large molecule
  • size exclusion support comprises HEC and Epi
  • the cationic moiety is N,N-diethylethylenediamine; wherein greater than 50% of the at least one negatively charged molecule is separated and greater than 90% of the of the at positively charged large molecule is recovered as flow-through.
  • the one or more molecules comprises at least one negatively charged small molecule and at least one positively charged large molecule
  • size exclusion support comprises HEC and Epi
  • the cationic moiety is diaminopentane
  • the equilibration buffer is Tris buffer
  • the negatively charged molecule is bovine serum albumin and the cationic moiety has binding capacity of 1.6 mg/mL for bovine serum albumin when the cationic moiety is diaminopentane, and the equilibration buffer is Tris buffer.
  • the negatively charged molecule is bovine serum albumin and the cationic moiety has binding capacity of 3.11 mg/mL for bovine serum albumin when the cationic moiety is polyethylene imine, and the equilibration buffer is Tris buffer.
  • the one or more molecules comprises at least one negatively charged small molecule and at least one positively charged large molecule
  • size exclusion support comprises HEC
  • the cationic moiety is polyethylene imine
  • the equilibration buffer is Tris buffer
  • kits for separating a positively charged large molecule from one or more negatively charged small molecules in a sample comprising: a porous size exclusion support having at least one cationic moiety associated therewith, wherein the cationic moiety can associate with and capture the at least one negatively charged small molecule; and instructions for using the porous size exclusion support.
  • the kit further comprises an equilibration buffer.
  • the equilibration buffer is Tris buffer or triethylammonium bicarbonate.
  • the kit further comprises a system comprising: a container housing the porous size exclusion support; and a receptacle positioned to receive flow-through the support.
  • the system of the kit is configured to operate by gravity flow, a centrifugal force, a positive pressure, a negative pressure, vacuum, and combinations thereof.
  • the container of the kit is a spin column, a multi-well filter plate, or a multi-well plate.
  • Matrixes comprising at least one size exclusion support and at least one cationic moiety for separating and/or extracting one or more small molecules from a sample that can associate with the one or more small molecules using differences in one or more properties, such as but not limited to, the size of the molecules, the charge of the molecules, the isoelectric point of the molecules, and/or any combination of these properties were prepared and tested.
  • exemplary size exclusion supports were modified with cationic moieties comprising functional groups that can associate with negatively charged small molecules by either ionic interaction, hydrophilic interaction, or any other interaction(s) that can associate with and thereby remove negatively charged small molecules from a sample while allowing at least one large positively charged molecules in the sample to be excluded and collected.
  • BSA removal and IgG purification kits were used.
  • the Abcam BSA Removal Kit (ab173231) commercially sold by Abcam and kits comprising a Melon Gel resin used for binding and removing serum proteins such as the MelonTM Gel IgG Purification Kit commercially sold by Thermo ScientificTM were used in the following examples.
  • crosslinked agarose beads bound to Cibacron Blue F3GA dye such as the Affi-Gel® Blue Gel commercially sold by Bio-Rad was used in the following examples.
  • vicinal diols located on these size exclusion support columns, were oxidized using a periodate to generate aldehyde groups.
  • PDA, branched PEI, or DEED were prepared in PBS, the pH was adjusted to a range of 8.0-8.5, and reacted with the oxidized columns according to the following procedure.
  • Different Size Exclusion Support's see Table 1) with different MWCOs were reacted with cationic reagents PDA and DEED to produce the Multimodal Resins of Table 2 according to Scheme 2.
  • Scheme 2 involves the following steps: (1) 100 mL's of size exclusion base matrix resin were prepared; (2) 2.3 grams of sodium metaperiodate were dissolved in water to prepare 100 mL's of 23 mg sodium metaperiodate/mL of resin; (3) adding 100 mL's of 23 mg sodium metaperiodate mL of resin to the 100 mL's of size exclusion matrix resin bed; (4) the reaction was allowed to proceed for 4 hours at room temperature with constant overhead stirring, thereby oxidizing vicinal diols present in the HEC to aldehyde groups; (5) preparing 100 mL volume of 50-150 mg/mL concentrations of the cationic moiety such as, but not limited to, PDA, PEI, or DEED in PBS and adjusting the pH of solution to be in the range of 8.0-8.5; (6) adding the prepared reagent to the resin slurry, whereby the amines provided by the reagents react with the aldehyde groups formed in the periodate oxidizing (step
  • Resin MWCO Effect on BSA Removal and Antibody Recovery In this example, the MWCO effect of the Resin A embodiment, the Resin B embodiment, the Resin D embodiment, the Resin E embodiment, the Resin F embodiment of Table 2 were compared to determine the effect of increasing the MWCO on the removal of BSA from a BSA (2 mg/mL) and IgG (2 mg/mL) mixture.
  • FIG. 9 is an image of a gel showing the removal of BSA from the BSA/IgG described above and in Table 2.
  • the BSA removal property which is indicated by lower band disappearance in the gel, increased in the following order respectively: the Resin F embodiment (45 kDa MWCO)>the Resin D embodiment (40 kDa MWCO)>the Resin E embodiment (30 kDa MWCO)>the Resin B embodiment (7 kDa MWCO)>the Resin A embodiment (2 kDa MWCO).
  • the MWCO in addition to the PDA modified embodiments had a significant effect on removing BSA from a sample.
  • MGPB Melon Gel Purification Buffer
  • Tris Buffer A positively charged buffer with no negative charge. Without being bound by this theory, the absence of a negative charge will allow for the BSA binding capacity to increase, similar to MGPB; however, also repel positively charged amine groups on IgG and thereby increase the IgG recovery.
  • FIG. 10 is an image of a gel showing the BSA removal and IgG recovery according to Table 3.
  • Lane 1A and Lane 1B show no BSA removal, however, the PDA modified resin embodiments removed more BSA from the BSA-IgG mixture. Moreover, the concentration of PDA (50 mg/mL versus 150 mg/mL) had little effect on BSA removal and the 40 kDa MWCO resin embodiments in Lanes 4A and 4B showed a lower capacity to remove BSA than the 45 kDa MWCO resin embodiments in Lanes 3A and 3B. Furthermore, the buffer used for equilibration demonstrated that the resin embodiments equilibrated with MGPB exhibited a lower IgG recovery than resin embodiments equilibrated with 50 mM Tris.
  • FIG. 11 is an image of a gel showing the removal of BSA and IgG recovery for a sample comprising a mixture of BSA (10 mg/mL) and IgG (1 mg/mL) by the Resin G embodiment (as described in Table 1, provided herein) equilibrated with 50 mM Tris pH 7 (Lane 1), 50 mM TEAB pH 5 (Lane 2), 50 mM TEAB pH 7 (Lane 3), 50 mM sodium acetate pH 5 (Lane 4), 50 mM sodium acetate pH 7 (Lane 5), 50 mM HEPES pH 5 (Lane 6), 50 mM HEPES pH 7 (Lane 7), 1 mg/mL rabbit IgG/10 mg /mL BSA (Lane 8), 1 mg/mL rabbit IgG, (Lane 9), 10 mg/mL BSA.
  • the BSA binding capacity (300 ⁇ L of 10 mg/mL) and the IgG recovery (2 mg/mL) of the Resin 5 embodiment of Table 1 modified with 150 mg/mL PEI was compared to the Resin 3 embodiment of Table 1 modified with 150 mg/mL PEI, Affi-Gel® Blue Gel (Bio-Rad), and the MelonTM Gel IgG Purification Kit (Thermo ScientificTM) in a sample comprising 300 ⁇ L of 10 mg/mL.
  • FIG. 12 is a bar graph showing the mg BSA bound/mL resin.
  • the Resin 5 embodiment (as described in Table 1, provided herein) had a binding capacity of 3.11 mg BSA bound/mL resin
  • the Resin 3 embodiment (as described in Table 1, provided herein) had a binding capacity of 2.45 mg BSA bound/mL resin
  • the MelonTM Gel IgG Purification Kit had a binding capacity of 1.26 mg BSA bound/mL resin
  • the Affi-Ge® Blue Gel Bio-Rad
  • FIG. 13 is a bar graph showing the IgG recovery (2 mg/mL). Accordingly, the Resin 5 embodiment (as described in Table 1, provided herein) exhibited a 72% volume recovery, the Resin 3 embodiment (as described in Table 1, provided herein) exhibited a 61% volume recovery, the MelonTM Gel IgG Purification Kit exhibited an 80% volume recovery, and the Affi-Gel® Blue Gel (Bio-Rad) exhibited a 25% volume recovery.
  • this example demonstrates that desirable BSA binding capacity was achieved when PEI was used as the modification reagent, had a comparable BSA binding capacity to the Affi-Gel® Blue Gel (Bio-Rad), and comparable IgG recovery to the MelonTM Gel IgG Purification Kit.
  • the Resin 5 embodiments of Table 1 were modified with different concentrations of PDA to produce the Resin F embodiment and the Resin K embodiment as described in Table 2, which were compared to commercially available Abcam BSA Removal Kit and MelonTM Gel IgG Purification Kit (Thermo ScientificTM) for their ability to remove BSA and recovery IgG from a sample comprising a BSA-IgG mixture.
  • Table 4 presents the data of FIG. 14 A , which is an image of a gel showing the BSA removal capacity and IgG recovery capacity of the Resin 5 embodiments modified with different concentrations of PDA to produce the Resin F embodiment and the Resin K embodiment versus Abcam BSA Removal Kit and MelonTM Gel IgG Purification Kit (Thermo ScientificTM).
  • Lane 5 is the BSA 10 mg/mL and GAR 1 mg/mL mixture
  • Lane 6 is the 1 mg/mL GAR
  • Lane 7 is the BSA 10 mg/mL
  • all lanes were normalized and samples were loaded at 10 ⁇ L/well on the gel.
  • FIG. 14 A shows superior BSA removal IgG recovery properties in Lane 1 and Lane 2 when compared to commercially available Abcam BSA Removal Kit and the MelonTM Gel IgG Purification Kit (Thermo ScientificTM) in Lane 3 and Lane 4 respectively.
  • FIG. 14 B is the is a bar graph obtained from quantitating the bands from FIG. 14 A using IBright image analysis software. Accordingly, the Resin F embodiment (Resin 5 embodiment of Table 1 modified with 150 mg/mL PDA) had a 85.7% IgG recovery and a 98.8% BSA removal; Resin K (Resin 5 embodiment of Table 1 modified with 75 mg/mL PDA) showed an 86.7% IgG recovery and an 82.6% BSA removal; Abcam BSA Removal Kit had a 53.3% IgG recovery and a 65.78% BSA removal; and MelonTM Gel IgG Purification Kit (Thermo ScientificTM) had a 78.7% IgG recovery and a 8.3% BSA removal. Therefore, the Resin F embodiment and the Resin K embodiment achieved a higher IgG recovery and BSA removal than the Abcam BSA Removal Kit and the MelonTM Gel IgG Purification Kit (Thermo ScientificTM).
  • FIG. 15 is a bar graph showing recovery percentage at 42,000 Da, 67,000 Da, 80,000 Da, and 150,000 Da for the Resin 5 embodiment, the Resin 6 embodiment, the Resin 7 embodiment, and the Resin 8 embodiment.
  • the Resin 5 embodiment exhibited an 86% at 42,000 Da, 94% recovery at 67,000 Da, 92% recovery at 80,000 Da, and 94% recovery at 150,000 Da.
  • the Resin 6 embodiment exhibited an 82% recovery at 42,000 Da, 84% recovery at 67,000 Da, 90% recovery at 80,000 Da, and 92% recovery at 150,000 Da.
  • the Resin 7 embodiment exhibited a 66% recovery at 42,000 Da, 76% recovery at 67,000 Da, 84% recovery at 80,000 Da, and 86% recovery at 150,000 Da.
  • the Resin 8 embodiment exhibited a 58% recovery at 42,000 Da, 75% recovery at 67,000 Da, 75% recovery at 80,000 Da, and 83% recovery at 150,000 Da.
  • the Resin F embodiment, the Resin G embodiment, the Resin H embodiment, Resin I embodiment, and Resin J were produced according to Table 2 and were tested for their ability to remove BSA and recover GAR from a sample comprising a BSA-GAR mixture and compared to commercially available MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit.
  • FIG. 16 A is an image of a gel showing the BSA removal and antibody recovery and Table 5 is a key of FIG. 16 A .
  • the Resin F embodiment (45 K MWCO) and Resin G embodiment (50 K MWCO) showed the most BSA removal and IgG recovery from the sample comprising the BSA (10 mg/mL) and GAR IgG (1 mg/mL) mixture when compared to the MeloTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® (Bio-Rad) and Abcam BSA Removal Kit.
  • Resin H embodiment (80 K MWCO) and the Resin I embodiment (90 K MWCO), which were modified with 75 mg/mL PDA performed better in removing BSA and recovering IgG when compared to the MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit; however, did not perform as well in recovering IgG than the Resin F embodiment and the Resin G embodiment.
  • Resin J was produced by reacting the Resin 6 embodiment of Table 1 with PDA and then was further reacted the open amine end of PDA with dextran having a molecular weight of 1,000,000; however, this did not improve the performance in comparison to Resin F and G.
  • FIG. 16 B is a bar graph further demonstrating the GAR (1 mg/mL) recovery and BSA (10 mg/mL) removal.
  • the Resin F embodiment showed a 83% GAR recovery and a 99% BSA removal
  • the Resin G embodiment showed a 93% GAR recovery and a 100% BSA removal
  • the Resin H embodiment showed a 63% GAR recovery and a 99% BSA removal
  • the Resin I embodiment showed a 76% GAR recovery and a 95% BSA removal
  • the Resin J embodiment showed a 82% GAR recovery and a 100% BSA removal
  • MelonTM Gel IgG Purification Kit (Thermo ScientificTM) showed a 106% GAR recovery and a 82% BSA removal
  • Affi-Gel® Blue Gel showed a 73% GAR recovery and a 65% BSA removal
  • Abcam BSA Removal Kit showed a 125% GAR recovery and a 85% BSA removal.
  • Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment, and Resin J embodiment were produced according to Table 2 and compared to MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit for their ability to remove BSA and IgG from a sample comprising a mixture of BSA (10 mg/mL) and GAR IgG (0.1 mg/mL).
  • FIG. 17 is an image of a gel showing the BSA removal and IgG recovery and Table 6 is a key of FIG. 17 .
  • the Resin F embodiment and the Resin G embodiment showed the best BSA removal and IgG recovery from the mixture comprising BSA (10 mg/mL) and GAR IgG (0.1 mg/mL), when compared to MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. Furthermore, Resin H performed better than MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit; however, did not perform as well in recovering IgG than the Resin F embodiment and the Resin G embodiment.
  • the Resin I embodiment showed a lower ability to remove BSA when the sample comprised a 0.1 mg/mL GAR and 10 mg/mL BSA mixture.
  • the Resin J embodiment did not perform better than the Resin F embodiment or the Resin G embodiment.
  • the MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit performed poorly in removing BSA and recovering GAR IgG in sample comprising a low GAR-BSA mixture (0.1 mg/mL GAR and 10 mg/mL BSA.
  • Lane 11 shows the BSA (10 mg/mL) only, which shows the smear obtained by running only BSA.
  • a mixture of GAR (1 mg/mL) and BSA (10 mg/mL) was passed and treated through the Resin F embodiment according to Table 2, MelonTM Gel IgG Purification Kit (Thermo ScientificTM), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit.
  • the resulting flow-through was then labeled with NHS DyLightTM 488 (Thermo ScientificTM) and the free dye was cleaned up using PierceTM Dye and Biotin Removal Resin.
  • FIG. 18 shows the fluorescent dye labeling to BSA removed from the antibody before the labeling reaction and Table 7 shows a key corresponding to FIG. 18 .
  • FIG. 18 shows the efficiency of labeling to the GAR when BSA was removed as demonstrated by the Resin F embodiment versus the MelonTM Gel IgG Purification Kit (Thermo ScientificTM) and Affi-Gel® Blue Gel (Bio-Rad) in which the labeling was comprised due to the unremoved BSA.
  • Abcam BSA Removal Kit demonstrated comparable results with the Resin F embodiment.
  • FIG. 19 is an image of the fluorescent dye labeling to BSA removed from the antibody before labeling the reaction.
  • a mixture of GAR (0.1 mg/mL) and BSA (10 mg/mL) was passed through the Resin F embodiment according to Table 2, the Resin G embodiment according to Table 2, MelonTM Gel IgG Purification Kit (Thermo ScientificTM) , Affi-Gel® Blue Gel (Bio-Rad), and BSA Removal Kit.
  • the resulting flow-through was then labeled with NHS DyLightTM 650 (Thermo ScientificTM) and the free dye cleaned up using PierceTM Dye and Biotin Removal Resin.
  • Table 8 is a key of FIG. 19 .
  • FIG. 19 demonstrates the efficiency of labeling to the GAR when BSA was removed as demonstrated by the Resin F embodiment and the Resin G embodiment when compared to MelonTM Gel IgG Purification Kit, Affi-Gel® Blue Gel (Bio-Rad), Abcam BSA Removal Kit in which the labeling efficiency was comprised due to unremoved BSA.
  • FIG. 19 indicates that even at low antibody concentrations such as 0.1 mg/mL, the Resin F embodiment and the Resin G embodiment were able to recover the antibody after the BSA was cleaned up and were able to successfully dye label it.
  • FIG. 20 is an image of a gel showing the results of the Resin F embodiment and the Resin G embodiment versus the start mixture where Lanes 1, 5, and 9 comprise the rabbit serum, Lanes 2, 6, 10 comprise the mouse serum, Lanes 3, 7, 11 comprise the human plasma, and Lanes 4, 8, and 12 comprise the human serum; and Table 9 shows the data of FIG. 20 .
  • the Resin F embodiment and the Resin G embodiment demonstrated an excellent ability to remove albumin and a good ability in recovering IgG from different serum species.
  • the starting amount and the flow-through amounts were measured at A 280 using a nanodrop.
  • the A 280 amount was calculated by measuring the measuring the A 280 on the nanodrop and taking the volume of the sample added and recovered into account.
  • a 280 measurement of BSA at 10 mg/mL was determined using the Nanodrop as the control.
  • FIG. 21 A is a bar graph showing the A 280 amount of the rabbit IgG A 280 amount.
  • the Resin G embodiment equilibrated with 50 mM Tris (pH 7.0) had a 123 A 280 amount; the Resin G embodiment equilibrated with 50 mM Tris (pH 5.0) had a 124 A 280 amount; the resin G embodiment equilibrated with 50 m Tris (pH 7.0 +stacker 20 ⁇ L) had a 139 A 280 amount;
  • the BSA-rabbit IgG start mixture had a 824 A 280 amount;
  • the Rabbit IgG start had a 133 A 280 amount; and the BSA only had a 634 A 280 amount.
  • FIG. 21 B is a bar graph showing the A 280 amount of the GAR.
  • the Resin G embodiment equilibrated with 50 mM Tris (pH 7.0) had a 97 A 280 amount; the Resin G embodiment equilibrated with 50 mM Tris (pH 5.0) had a 101 A 280 amount; the resin G embodiment equilibrated with 50 m Tris (pH 7.0 +stacker 20 ⁇ L) had a 99 A 280 amount; the BSA-GAR start mixture had a 834 A 280 amount; the GAR start had a 100 A 280 amount; and the BSA only had a 634 A 280 amount.
  • FIG. 22 is an image of a gel showing the removal of BSA from the primary antibody GAPDH before conjugating it with DyLightTM 650 (Thermo ScientificTM) and Table 10 shows the data of FIG. 22 .
  • the resin was spun at 3000 ⁇ G and 6000 ⁇ G. 1 mg/mL GAPDH was spiked with 10 mg/mL BSA and passed through the Resin G embodiment according to Table 2. The flow-through was collected and the resin was washed with 50 mM Tris to collect the GAPDH that may have been bound to the resin. The flow-throughs from the two elution's were collected, pooled, then conjugated with NHS DyLightTM 650 (Thermo ScientificTM) and cleaned using PierceTM Dye and Biotin Removal Resin. GAPDH with BSA was also labeled and Free Dy 650 were added as control lanes was also labeled.
  • This example demonstrates successful removal of BSA from primary antibody GAPDH before conjugating with DyLightTM 650 (Thermo ScientificTM) and thus demonstrates complete removal of BSA for spin speeds at 3,000 ⁇ G and 6,000 ⁇ G.
  • the ability the Resin G embodiment according to Table 2 to remove BSA from primary antibody Calreticulin before conjugating it with DyLightTM 680 NHS (Thermo ScientificTM) was tested. 1 mg/mL Calreticulin antibody was obtained with 1 mg/mL BSA added as a stabilizer. This antibody was passed through the Resin G embodiment and the flow-through was collected and the resin was washed three times with 50 mM Tris to collect the primary antibody that may have been bound to the resin. The flow-throughs were collected, pooled, concentrated, and then conjugated with DyLightTM 680 NHS (Thermo ScientificTM) and cleaned using PierceTM Dye and Biotin Removal Resin.
  • FIG. 23 is an image a gel obtained by loading the flow-throughs and stained using Coomassie stain using Pierce Power blotter. Table 11 shows the data of FIG. 23 .
  • Lane 1 shows the Calreticulin that is free from BSA after passing through the Resin G embodiment.
  • Lane 2 shows the Calreticulin that is conjugated to the DyLightTM 680 (Thermo ScientificTM) after BSA is removed.
  • Lane 3 is the Calreticulin as received with BSA added as a stabilizer. In view of this, this example demonstrates the successful removal of BSA from the primary antibody Calreticulin before conjugating it with DyLightTM 680 (Thermo ScientificTM) by the Resin G embodiment.
  • the membrane was washed and then incubated with HSP 90 primary antibody.
  • the membrane was washed, then incubated with GAR Dy 650, washed and then scanned on IBright imager using the fluorescence mode.
  • GAR conjugated with Dy 650 without BSA removal was used as a control for comparison.
  • FIG. 24 A is an image of a gel showing the BSA removed from GAR (left) and the BSA not removed from GAR (right).
  • FIG. 24 B is a bar graph showing the fluorescence intensity of the removed BSA and the unremoved BSA of the HeLa lysate load. As shown in FIG.
  • HeLa lysate load for the BSA removed at 10 ⁇ g had a fluorescence intensity of 13,000,000 and the unremoved BSA had a fluorescence intensity of 4,000,000; HeLa lysate load for the BSA removed at 5 ⁇ g had a fluorescence intensity of 9,000,000 and the unremoved BSA had a fluorescence intensity of 3,800,000; HeLa lysate load for the BSA removed at 2.5 ⁇ g had a fluorescence intensity of 7,800,000 and the unremoved BSA had a fluorescence intensity of 3,800,000; and HeLa lysate load for the BSA removed at 1.25 ⁇ g had a fluorescence intensity of 4,100,000 and the unremoved BSA had a fluorescence intensity of 1,800,000.

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